This invention relates to recombinant-DNA-technology. Specifically this invention relates to new recombinant yeast strains transformed with xylose reductase and/or xylitol dehydrogenase enzyme genes. A yeast strain transformed with the xylose reductase gene is capable of reducing xylose to xylitol and consequently of producing xylitol in vivo. If both of these genes are transformed into a yeast strain, the resultant strain is capable of producing ethanol on xylose containing medium during fermentation.
Further, the said new yeast strains are capable of expressing the said two enzymes. Xylose reductase produced by these strains can be used in an enzymatic process for the production of xylitol in vitro.
Xylose Utilization
Xylose appears in great abundance in nature. It can constitute as much as 40% of a lignocellulosic material (Ladisch et al., 1983). By fermentation xylose can be converted to ethanol which can be used as a liquid fuel or a chemical feedstock. Enzymatically or as a by-product of fermentation xylose can also be converted to xylitol which is a promising natural- sweetener having dental caries reducing properties. Xylitol can also be used by diabetics. For the production of ethanol which is a cheap product it is important that the raw material can be fermented directly with as little pretreatment as possible. For the production of xylitol which is meant for human consumption it is important that the process involves GRAS organisms.
Natural xylose utilizers are found among bacteria, yeast and fungi. In all organisms xylose is converted to xylulose which is phosphorylated to xylulose-5-phosphate (X5P) with xylulokinase. X5P then enters the Embden-Meyerhof pathway (glycolysis) via the pentose phosphate shunt.
Bacteria like Escherichia coli, Bacillus sp., Streptomyces sp. and Actinoplanes sp. convert xylose directly to xylulose with a xylose isomerase (XI). Thus bacteria do not produce xylitol as an intermediate during xylose utilization. Those which ferment xylose to ethanol do so with poor yields because a number of by-products are also produced (Skoog and Hahn-Hxc3xa4gerdal, 1988). In xylose utilizing yeasts such as Pichia stipitis, Candida shehatae and Pachysolen tannophilus this reaction occurs in two steps: first xylose is reduced to xylitol with a xylose reductase (XR) and the xylitol is oxidized with a xylitol dehydrogenase (XDH) to xylulose.
Pure xylose solutions are fermented with high yields and good productivities by xylose utilizing yeasts such as P. stipitis, C. shehatae and P. tannophilus (Slininger et al., 1987; Prior et al., 1989). However, they do not generally survive in the hostile environment of an untreated raw material such as eg. spent sulphite liquor or hydrogen fluoride-pretreated and acid-hydrolyzed wheat straw (Lindxc3xa9n and Hahn-Hxc3xa4dgerdal, 1989). The one exception, P. tannophilus, produces mainly xylitol and glycerol in response to this environment. In order to efficiently ferment such raw materials with the xylose utilizing yeasts such as P. stipitis, C. shehatae and P. tannophilus the raw material has to undergo expensive pretreatments with ion-exchange resins (Clark and Mackie, 1984) or steam stripping (Yu et al., 1987).
Saccharomyces cerevisiae, bakers"" yeast, ferments spent sulphite liquor or hydrogen fluoride-pretreated and acid-hydrolyzed wheat straw to ethanol (Lindxc3xa9n and Hahn-Hxc3xa4gerdal, 1989). S. cerevisiae cannot utilize xylose efficiently and cannot grow on xylose as a sole carbon source. In the presence of the bacterial enzyme xylose isomerase, which converts xylose to xylulose, S. cerevisiae can, however, ferment both pure xylose solutions (Hahn-Hxc3xa4gerdal et al., 1986) and untreated raw materials (Lindxc3xa9n and Hahn-Hxc3xa4gerdal, 1989a,b) to ethanol with yields and productivities that are in the same order of magnitude as those obtained in hexose fermentations.
Similar results have been obtained with Schizosaccharomyces pombe (Lastick et al., 1989). Thus, both S. cerevisiae and Sch. pombe have a functioing xylulokinase enzyme. It has also been found that S. cerevisiae can take up xylose (Batt et al., 1986; van Zyl et al., 1989; Senac and Hahn-Hxc3xa4gerdal, 1990).
Gong (1985) discloses a process for obtaining ethanol directly from D-xylose by xylose fermenting yeast mutants. According to Gong a parent yeast strain is selected (e.g. Candida sp. or Saccharomyces cerevisiae), which originally may have the ability to utilize D-xylose, and this parent strain is then exposed e.g. to UV-radiation so as to induce mutation. However, no information about the reason why the mutants obtained are able to utilize xylose, is given in the reference. Further, Gong did not introduce any new coding and/or regulatory sequences to said strains by genetic engineering techniques to enhance xylose fermentation.
Xylitol is industrially manufactured at the moment by chemical reduction of hemi-cellulose hydrolysates. Poisoning of the expensive catalyst used in the reduction step and formation of side-products difficult to be separated from the end product are the main problems in this process.
In literature there are numerous examples of microbiological methods to produce xylitol from pure xylose (eg. Onishi and Suzuki, 1966; Barbosa et al., 1988). Best producers in this method are yeasts especially belonging to the Candida-genera. Also some bacteria such as Enterobacter (Yoshitake et al., 1973a) and Corynebacterium species (Yoshitake et al., 1973b) and some molds eg. Penicillium chrysogenum (Chiang and Knight, 1960) produce xylitol from pure xylose.
In a microbiological method describing the best yields of xylitol production (Ojamo et al., 1987) Candida guilliermondii yeast is cultivated under strictly controlled aeration in a xylose containing medium either as a batch or a fed-batch process. Xylitol yields 50-65% were obtained. The yield could be increased to 76% by adding furfuraldehyde to the cultivation medium.
Cell-free extracts from Candida pelliculosa (xylose reductase) and Methanobacterium sp. (hydrogenase, F420, NADP, F420/(NADP oxidoreductase) has been used to produce xylitol in a membrane reactor with 90% conversion (Kitpreechavanich, 1985). With a cell-free extract from a Corynebacterium species 69% conversion has been obtained when 6-phosphogluconate was used for regeneration of the cofactor.
It has been shown that glucose dehydrogenase from B. megaterium has suitable properties as a NADPH regenerating enzyme (Kulbe et al., 1987). Thus gluconic acid from glucose can be produced simultaneously with xylitol in the enzymatic process. For the retention of the enzymes and the cofactor one can use ultrafiltration membranes. Cofactor retention may be achieved by the use of a derivatized cofactor having high enough molecular weight for the retention (Kulbe et al., 1987) or better by using negatively charged ultrafiltration membranes (Kulbe et al., 1989).
Attraction to use an enzymatic method is based on the possibility to use impure xylose containing raw materials which in the microbiological methods would inhibit the metabolism of the microbe used. Also the yields of xylitol are higher than in the microbiological methods with natural strains. On the other hand any microbiological method is more simple in large-scale practice at the moment.
The natural xylose utilizing yeasts such as P. stipitis, Candida sp. and P. tannophilus are not suitable for the production of either ethanol or xylitol for several reasons. The fermentation to ethanol requires pretreatment of the raw material which is cost-prohibitive for a cheap end-product such as ethanol. These species also lack the GRAS-status. Thus xylose utilization would most suitably be based on the use of baker""s yeast which has a GRAS-status.
In order to make S. cerevisiae an efficient xylose utilizer for the production of xylitol and ethanol an efficient enzyme system for the conversion of xylose to xylitol and xylulose should be introduced into this yeast. For the production of ethanol from xylose the XI genes from E. coli (Sarthy et al., 1987), B. subtilis and Actinoplanes missouriensis (Amore et al., 1989) have been cloned and transformed into S. cerevisiae. The XI protein made in S. cerevisiae had very low (1/1000 of the enzyme produced in bacteria) or no enzymatic activity. Thus, for some reasons the bacterial enzyme can not be made functional in yeast. Another possibility would be to transfer into S. cerevisiae the genes encoding XR and XDH from another yeast. The enzymes of P. stipitis should be good candidates in the light of the efficient utilization of pure xylose solutions discussed above. It can be anticipated that enzymes from another yeast would function better than bacterial enzymes when expressed in yeast. In addition, xylitol and ethanol could be produced with the same system and the system would combine the good xylose utilization of P. stipitis with resistance to inhibitors and general acceptance of S. cerevisiae. 
The present invention describes the isolation of genes coding for xylose reductase (XR) and xylitol dehydrogenase (XDH) from certain yeasts having these genes, the characterization of the genes and their transfer into, and their expression in Saccharomyces cerevisiae. 
This invention thus provides new recombinant yeast strains expressing xylose reductase and/or xylitol dehydrogenase enzymes.
The yeast strains according to the invention being transformed with the XR gene are capable of reducing xylose to xylitol in vivo. Xylose reductase produced by the new yeast strains according to the invention is also used in an enzymatic process for the production of xylitol in vitro.
The present invention further provides new yeast strains transformed with both of the above mentioned two genes. The coexpression of these genes renders the strain capable of fermenting xylose to ethanol from pure xylose solution or xylose containing solutions such as lignocellulosic hydrolyzates.